Antigen-specific immune responses are mediated by antigen-specific effector B and T lymphocytes. These cells originate from generally low frequency resting precursor cells expressing receptors for various antigens representing the whole repertoire and which, upon encounter with specific antigens and appropriate costimulation, become activated, expand and differentiate into effector cells.
Development of ex vivo immunotherapy for conditions such as cancer or viral infections is limited by the low frequency of antigen-specific precursor lymphocytes. For instance, virus-specific CTL precursor (CTLp) frequencies in the peripheral lymphoid tissues of mice are generally lower than 1/100,000–1/1,000,000 (Lau et al., 1994; Hou et al., 1994). Isolation of antigen-specific lymphocytes by capture on an antigen-coated support has been described for mouse spleen resting B cells specific for TNP (Snow et al, 1983a). The isolation procedure involved a rosetting step on haptenated horse red blood cells and allowed the recovery of hapten-specific B cells with a 40% purity. This technique has been useful to study the requirements for activation (Stein et al, 1986) as well as the initial signaling events following activation (Snow et al, 1986; Myers et al, 1987; Grupp et al, 1987; Noelle and Snow, 1990; Gold and DeFranco, 1994). However, this was a very favorable situation because of the relatively high frequency of B cells specific for TNP (about 1%) (Snow et al,1983a). No study on B cell activation using resting B cells specific for another antigen with low precursor frequency has been reported to date (Radbruch and Recktenwald, 1995).
Low precursor frequency is also a problem with T cells. Additionally, while B cells recognize the antigen directly, T cells recognize a complex structure made of the combination of an antigenic peptide bound to a major histocompatibility complex (MHC) molecule. TCR/MHC-peptide interaction has a low to moderate affinity (10−4–10−7 M range: Matsui et al, 1991; Weber et al, 1992; Sykulev et al, 1994a; Corr et al, 1994; Sykulev et al, 1994b). Antibodies usually exhibit affinities several orders of magnitude higher and exploit multivalency. New techniques of isolation of rare cell populations are available now. These are based on cell sorting and/or magnetic separation (Bellone et al, 1995; Radbruch and Recktenwald, 1995). Also, recombinant ligands for TCR are now available by combining recombinant empty MHC molecules (Jackson et al, 1992) and MHC-binding antigenic peptides (Engelhard, 1994; Ramensee et al, 1995). These synthetic MHC-peptide complexes can be immobilized on beads to yield multivalent ligands for the TCR. Theoretically, multivalency should help to overcome low affinity. The interaction between TCR and immobilized peptide-MHC complex has been previously shown to lead to the establishment of stable interactions in certain in vitro systems. First, MHC class I antigens immobilized on lipid monolayers (Nakanashi et al, 1983) or on lipid-coated cell-sized beads (Kane et al, 1988) are sufficient to cause binding of cloned allogeneic cytotoxic T cells (CTL). Second, syngeneic cloned CTL bind to MHC-coated beads in a peptide dependent manner (Kane and Mescher, 1993; Mescher, 1995). Third, a cloned syngeneic CTL can form aggregates with RMA-S cells, a cell line which expresses large amounts of empty MHC molecules, in a peptide specific manner (De Bruijn et al, 1992). In two of these reports, TCR-MHC-peptide interactions were not the primary mediator of adhesion. They rather played an initial role in the early events of aggregation, presumably by transducing signals that led to activation of adhesion via accessory molecules.
Here, we describe a method to isolate antigen-specific T cells using empty MHC class I molecules purified from Drosophila melanogaster cells (Jackson et al, 1992) immobilized on magnetic beads and loaded with peptide. This artificial substrate for T cells is coated with a high density of identical MHC-peptide complexes. T cell isolation was optimized using populations of naïve T cells purified from mice transgenic for the 2C TCR (Sha et al, 1988). Ligands of various affinities and specificities for the 2C TCR have been identified (Sykulev et al, 1994a, b). 2C T cells could be adsorbed on beads bearing peptide-MHC complexes which had an affinity for the 2C TCR as low as 10−4 M. Adsorption was MHC restricted and peptide specific since it occurred only with the proper MHC-peptide combinations recognized by the 2C TCR. Additionally, 2C T cells mixed with irrelevant T cells from a naive animal could be recovered using this adsorption procedure. This technique was successfully used to recover antigen-specific T cells from naive animals.